The quantative analysis of various metabolic components present in body fluids, e.g., glucose, bilirubin, urea nitrogen, uric acid, cholesterol, lactic acid dehydrogenation enzyme, creatine kinase, glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) is clinically important and is indispensable to the diagnosis of diseases, checking of medical treatment, and monitoring of prognosis. In the clinicochemical examination of blood or the like as a specimen, it is desirable that an examination with high accuracy be effected using only a small amount of a liquid specimen. A wet analysis process using a reagent solution has heretofore been used. However, such wet analysis process lacks rapidity.
A dry chemical analysis, i.e., a clinical analysis method using an analyzing reagent system incorporated in a water-permeable carrier in a substantially dry analysis element, such as, a test piece or multilayer analysis element, has been known. The dry chemical analysis process is better than the wet analysis process in ease of use, minimization of the amount needed of a specimen to be tested and rapidity of analysis In other words, the use of a dry multilayer analysis element enables a highly precise, simple and rapid analysis of a small amount of a liquid specimen Such a dry multilayer analysis element is disclosed in JP-B-53-21677 (The term "JP-B" as used herein means an "examined Japanese patent publication"), and JP-A-55-164356, and 60-222769. (The term "JP-A" as used herein means an "unexamined published Japanese patent application". Patents written in English and corresponding to Japanese Patent Publications cited in this application are shown hereinafter.).
A typical dry multilayer analysis element consists of a transparent support, a reagent layer, a reflective layer and a spreading layer. The reagent layer coated on a water impermeable transparent support (e.g., undercoated plastic film) comprises a reagent which reacts with the component to be determined contained in the liquid specimen and which developes color at an optical density corresponding to the amount of the component. The reflective layer is adapted to prevent light incident to the reagent layer from reaching the spreading layer so that the optical measurement of the reagent layer is not affected by the liquid specimen spotted on the spreading layer. The spreading layer is adapted to spread the spotted liquid specimen over an area substantially proportional to the amount of the liquid specimen When such a dry analysis element is used for quantitative determination, a liquid specimen, such as, whole blood is spotted onto the surface of the spreading layer in a specified amount The blood, thus spread over the spreading layer, passes through the reflective layer and reaches the reagent layer where it reacts with a reagent to develop color. After the liquid specimen is dropped on the spreading layer, the analysis element is kept at a predetermined temperature for a proper period of time to allow the color development reaction to proceed. The optical reflection density of the reagent layer is determined from the support side at a specified wavelength range. The quantitative determination is conducted from the optical reflection density on the basis of a predetermined calibration curve.
As described in JP-A-58-70163, if the reagent layer composed of a hydrophilic high molecular weight compound such as gelatin is used, a component having high molecular weight, such as protein (e.g., albumin), various enzymes which also are proteins, polysaccharides, or a hydrophobic component, such as cholesterol, triglyceride and bilirubin, cannot diffuse into the reagent layer, making it impossible to react with the reagent. Thus, such a component cannot be detected.
As a means for solving such a problem an approach has been taken which comprises allowing a high molecular weight component or hydrophobic component to undergo reaction in the spreading layer In accordance with this approach, these components are allowed to undergo reaction in the spreading layer without being diffused into the reagent layer. In one form of this process, a dye produced in the reagent layer and/or spreading layer is diffused through light reflecting layer into a detection layer provided below the light reflecting layer (near the support), and is then detected in the detection layer. In another form, a low molecular weight intermediate produced by the reaction in the spreading layer is diffused into a color reagent layer provided below the spreading layer (near the support) where it is detected. However, the former process is disadvantageous in that since the proportion of a dye diffused into the detection layer is low, the analysis sensitivity is insufficient. The latter is disadvantageous in that it is susceptible to interference from interfering components in the sample liquid. Examples of the former process include a process which comprises detection of a dye produced in the spreading layer without diffusing it into other layers In this process, layers other than the spreading layer are omitted. However, this process is disadvantageous in that it is difficult to analyze whole blood because the background is variable.
In order to enable the detection of a high molecular weight component or hydrophobic component, a dry multilayer analysis element comprising a reagent layer made of a grain structure comprising bound polymer grains has been proposed in JP-A-55-90859 and 58-70163. However, such an improved reagent layer exhibits certain disadvantage. If a light reflecting layer or the like provided between the spreading layer and the reagent layer inhibits the diffusion of a high molecular weight component or a hydrophobic component, the analysis of these components is made difficult.
As a light reflecting layer of a dry multilayer analysis element, a layer comprising grains of inorganic material, such as, titanium oxide or barium sulfate dispersed therein with a hydrophilic high molecular weight compound as a binder is disclosed in U.S. Pat. Nos. 3,992,158 and 4,042,335 (JP-B-58-18628). In such a layer, if the proportion (volume or weight) of the binder is too high, the permeation of a high molecular weight component or a hydrophobic component therethrough is difficult to achieve. On the contrary, if the proportion of the binder is too low, the mechanical properties of the layer are poor though permeation of a high molecular weight component or a hydrophobic component through the layer is possible. The layer becomes extremely brittle and is subject to cracking that may cause separation of the grains.